Programmed cell death, referred to as apoptosis, plays an indispensable role in the development and maintenance of tissue homeostasis within all multicellular organisms (Raff, Nature 356: 397-400, 1992). Genetic and molecular analysis from nematodes to humans has indicated that the apoptotic pathway of cellular suicide is highly conserved (Hengartner and Horvitz, Cell 76: 1107-1114, 1994). In addition to being essential for normal development and maintenance, apoptosis is important in the defense against viral infection and in preventing the emergence of cancer.
Considerable progress has been made in identifying molecules that regulate the apoptotic pathway at each level. Of note, both positive and negative regulators, often encoded within the same family of proteins, characterize the extracellular, cell surface and intracellular steps (Oltvai and Korsmeyer, Cell 79: 189-192, 1994).
The mitochondrion is a highly complex and compartmentalized organelle and is a prominent participant in apoptosis following a variety of death stimuli (Green and Reed, Science 281, 1309-1312, 1998; Kroemer et al., Annu. Rev. Physiol. 60, 619-642, 1998). The “multidomain” pro-apoptotic BCL-2 family members BAX and BAK prove necessary for the onset of mitochondrial dysfunction and cell death following remarkably diverse signals (Wei et al., Science 292, 727-730, 2001). Thus, mitochondria may prove an obligate organelle for apoptosis downstream of perhaps all intrinsic pathway signals. Release of cytochrome c from the intermembrane space (IMS) is a prominent facet of such intrinsic pathway deaths. Cytochrome c triggers a post mitochondrial pathway, forming an “apoptosome” of Apaf-1, cytochrome c and caspase-9 which subsequently cleaves the effector caspases-3,-7 (Li et al., Cell 91, 479-489, 1997).
The precise mechanism whereby cytochrome c is released across the outer mitochondrial membrane (OM) is less certain. Permeability transition (PT) that ultimately leads to mitochondrial swelling with secondary rupture of the OM and cytochrome c release has been noted in certain apoptotic and necrotic deaths (Lemasters et al., Biochim. Biophys. Acta 1366, 177-196, 1998). In its fully open conformation the PT pore (PTP), a high conductance inner membrane channel, is permeable to solutes up to 1500 Da (Bernardi, Physiol. Rev. 79, 1127-1155, 1999). However, openings of the PTP can also be transient and not cause swelling (Huser et al., Biophys. J. 74, 2129-2137, 1998; Petronilli et al., Biophys. J. 76, 725-734, 1999). As originally noted at the single channel level, the PTP flickers over milliseconds (msecs) between its open and closed states (Petronilli et al., FEBS Lett. 259, 137-143, 1989). Cyclosporin A (CsA) inhibits both activities of the PTP, presumably through its mitochondrial target cyclophilin D (Nicolli et al., J. Biol. Chem. 271, 2185-2192, 1996). Thus, models of cytochrome c release must also assess whether PT participates.
Defining the serial events responsible for cytochrome c release requires a distinct initiating event. The “BH3 domain-only” subset of BCL-2 members provides such a signal as they connect proximal death signals to the core apoptotic pathway at the mitochondria. The “BH3 domain-only” molecules BID, BAD, BIM, NOXA require the “multidomain” members BAX, BAK to release cytochrome c and induce cell death (Wei et al., 2001; Zong et al., Genes Dev. 15, 1481-1486, 2001; Cheng et al., Mol. Cell. 8, 705-711, 2001). For example, after CD95 (Fas) or TNFR1 engagement BID is cleaved by caspase-8 followed by N-myristoylation to induce its molecular activation (Luo et al., Cell 94, 481-490, 1998; Zha et al., Science 290, 1761-1765, 2000). Recombinant tBID (truncated p15 BID) is an ideal initiating event as it appears to function as a death ligand that induces the homo-oligomerization of BAK with subsequent release of cytochrome c from wild-type (wt) but not Bak-deficient mitochondria (Wei et al., Genes Dev. 14, 2060-2071, 2000). tBID releases cytochrome c without detectable swelling of the mitochondria (Shimizu and Tsujimoto, Proc. Natl. Acad. Sci. U.S.A. 97, 577-582, 2000; Eskes et al., J. Cell Biol. 143, 217-224, 1998; Wei et al, 2000) but increases the permeability of the OM (Kluck et al., J. Cell Biol. 147, 809-822, 1999).
Any model must also account for the rapid kinetics and complete extent of cytochrome c release (Goldstein et al., Nat. Cell Biol. 2, 156-162, 2000). High-voltage electron microscopic (HVEM) tomography of mitochondria has revealed that the IMS is very narrow, as the average distance between the OM and inner boundary membranes (IM) is only ˜20 nm (Frey and Manila, Trends. Biochem. Sci. 25, 319-324, 2000) consistent with functional estimates that only 15-20% of total cytochrome c is available in the IMS (Bernardi and Azzone, J. Biol. Chem. 256, 7187-7192, 1981). The pleomorphic, tubular cristae constitute highly sequestered compartments where the majority of oxidative phosphorylation complexes (Perotti et al., J. Histochem. Cytochem. 31, 351-365, 1983) and cytochrome c are located. Cristae junctions of ˜18 nm diameter physically separate the tubular cristae compartments from the narrow IMS in normal liver mitochondria. The major stores of cytochrome c (˜85%) are sequestered within the cristae, and computer modeling of this subcompartmentalization indicates ion and ADP diffusion gradients across the cristae junctions (Mannella et al., IUBMB Life, 52(3-5):93-100, 2001). A major challenge is to explain how this compartmentalized store of cytochrome c can be released in the absence of mitochondrial swelling. Therefore, investigating whether a structural reorganization occurs during apoptosis to mobilize the cristae stores of cytochrome c for release across the OM is desirable.
Some disease conditions are affected by the development of a defective apoptotic response. For example, neoplasias may result, at least in part, from an apoptosis-resistant state in which cell proliferation signals inappropriately exceed cell death signals. Furthermore, some DNA viruses such as Epstein-Barr virus, African swine fever virus and adenovirus, parasitize the host cellular machinery to drive their own replication and at the same time modulate apoptosis to repress cell death and allow the target cell to reproduce the virus. Moreover, certain disease conditions such as lymphoproliferative conditions, cancer including drug resistant cancer, arthritis, inflammation, autoimmune diseases and the like may result from a defect in cell death regulation. In such disease conditions, it would be desirable to promote apoptotic mechanisms.
Furthermore, in certain disease conditions it would be desirable to inhibit apoptosis such as in the treatment of immunodeficiency diseases, including AIDS, senescence, neurodegenerative diseases, ischemia and reperfusion, infertility, wound-healing, and the like. In the treatment of such diseases it would be desirable to diminish or inhibit cell death agonist activity.
Since there is an unmet need in regard to apoptotic modulation, it is desirable to identify novel proteins or critical protein domains which have cell-death agonist/antagonist properties and to utilize these as a basis for treatment modalities in advantageously modulating the apoptotic process in disease conditions involving either inappropriate repression or inappropriate enhancement of cell death.